In Drosophila, the gene product (Dlgl) of the tumor suppressor gene l(2)gl binds to nonmuscle myosin II (NMII) and the association is regulated by a kinase which binds to Dlgl (Kalmes et al., J. Cell Sci. 109, 1359, 1996). Mammalian cells contain a closely related protein which also binds a kinase regulating association with NMII. Since the kinase identity is unknown, we constructed a GST-fusion protein containing amino acids 617-731 of Mgl, the mouse homolog of Dlgl, to assist in purification of the kinase. The fusion protein contains 4 highly conserved serines, S650, 654, 658 and 662, which have been implicated as targets for the kinase. When GST-Mgl was incubated with PC12 lysates, precipitated with glutathione-agarose and subjected to in vitro kinase assay, the precipitates contained a tightly-bound kinase activity which phosphorylated GST-Mgl; mutation of the serine residues to alanine blocked phosphorylation. Taking advantage of the tight association of GST-Mgl and kinase, kinase activity was assayed in a pull-down assay using the fusion protein as affinity-ligand and substrate. Kinase activity was partially purified from a 25-60% ammonium sulfate fraction of rat brain high-speed supernatants. Sephacryl 300 chromatography was followed by ion exchange chromatography on Q-Sepharose, affinity chromatography on Blue Sepharose and gel filtration on Superose 6, leading to a 700-fold increase in specific activity. A major protein band of 80 kD in the extract binds to GST-Mgl and is phosphorylated on incubation with ATP; phosphorylation of both the fusion protein and the 80 kD band is inhibited by the protein kinase C (PKC) inhibitor GF109203X. Western blotting of proteins bound to GST-Mgl shows association of several PKC isoforms with the fusion protein. Future experiments will examine the binding of PKC isoforms to full-length Mgl and the phosphorylation of the full-length protein and its regulation in vitro and in vivo.